U.S. patent application number 16/486438 was filed with the patent office on 2020-04-09 for therapeutic agent for pulmonary fibrosis, ptprr expression-promotor, and kit for treatment of pulmonary fibrosis.
This patent application is currently assigned to ROHTO PHARMACEUTICAL CO., LTD.. The applicant listed for this patent is ROHTO PHARMACEUTICAL CO., LTD. Public University Corporation Nara Medical University. Invention is credited to Tetsuo Furuno, Toshihiro Ito, Hiroshi Kimura, Makiko Sawada, Masanori Yoshikawa.
Application Number | 20200108099 16/486438 |
Document ID | / |
Family ID | 63169871 |
Filed Date | 2020-04-09 |
United States Patent
Application |
20200108099 |
Kind Code |
A1 |
Furuno; Tetsuo ; et
al. |
April 9, 2020 |
THERAPEUTIC AGENT FOR PULMONARY FIBROSIS, PTPRR
EXPRESSION-PROMOTOR, AND KIT FOR TREATMENT OF PULMONARY
FIBROSIS
Abstract
The purpose of the present invention is to provide a novel
treatment agent for pulmonary fibrosis for which there is currently
no effective drug therapy established. The present invention is a
therapeutic agent for pulmonary fibrosis containing mesenchymal
stem cells. The pulmonary fibrosis is preferably usual interstitial
pneumonia (UIP) or idiopathic pulmonary fibrosis (IPF). Also, the
mesenchymal stem cells are preferably derived from adipose tissue
or are allogeneic.
Inventors: |
Furuno; Tetsuo; (Osaka,
JP) ; Ito; Toshihiro; (Nara, JP) ; Kimura;
Hiroshi; (Nara, JP) ; Yoshikawa; Masanori;
(Nara, JP) ; Sawada; Makiko; (Nara, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ROHTO PHARMACEUTICAL CO., LTD.
Public University Corporation Nara Medical University |
Osaka
Nara |
|
JP
JP |
|
|
Assignee: |
ROHTO PHARMACEUTICAL CO.,
LTD.
Osaka
JP
Public University Corporation Nara Medical University
Nara
JP
|
Family ID: |
63169871 |
Appl. No.: |
16/486438 |
Filed: |
February 9, 2018 |
PCT Filed: |
February 9, 2018 |
PCT NO: |
PCT/JP2018/004623 |
371 Date: |
August 15, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/28 20130101;
A61P 43/00 20180101; A61P 11/00 20180101; A61K 45/00 20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61P 11/00 20060101 A61P011/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 15, 2017 |
JP |
2017-025790 |
Claims
1-7. (canceled)
8. A method for treating pulmonary fibrosis, comprising
administration of an effective amount of mesenchymal stem cells to
a subject having pulmonary fibrosis.
9. The method according to claim 8, wherein the pulmonary fibrosis
is usual interstitial pneumonia (UIP) or idiopathic pulmonary
fibrosis (IPF).
10. The method according to claim 8, wherein the mesenchymal stem
cells are derived from an adipose tissue.
11. The method according to claim 9, wherein the mesenchymal stem
cells are derived from an adipose tissue.
12. The method according to claim 8, wherein the mesenchymal stem
cells are allogenic.
13. The method according to claim 9, wherein the mesenchymal stem
cells are allogenic.
14. The method according to claim 10, wherein the mesenchymal stem
cells are allogenic.
15. The method according to claim 11, wherein the mesenchymal stem
cells are allogenic.
16. A method for treating pulmonary fibrosis, comprising
administration of an effective amount of a PTPRR expression
promotor to a subject having pulmonary fibrosis.
17. The method according to claim 16, wherein the PTPRR expression
promotor dephosphorylates phosphorylated ERK.
Description
TECHNICAL FIELD
[0001] The present invention relates to a Therapeutic agent for
Pulmonary fibrosis, a PTPRR expression promotor, and a kit for
treatment of pulmonary fibrosis.
BACKGROUND ART
[0002] Idiopathic interstitial pneumonia (IIP) is a disease group
of interstitial pneumonias of unknown etiology that share similar
clinical features and classified into 6 histologic subtypes. All
the subtypes are characterized by varying degrees of inflammation
and fibrosis and all cause dyspnea and typical radiographic
abnormalities. The 6 histologic subtypes of idiopathic interstitial
pneumonia in decreasing order of frequency are usual interstitial
pneumonia (VIP), nonspecific interstitial pneumonia, bronchiolitis
obliterans with organizing pneumonia, interstitial lung disease
(ILD) with respiratory bronchiolitis, desquamative interstitial
pneumonia, and acute interstitial pneumonia.
[0003] Idiopathic pulmonary fibrosis (IPF, idiopathic fibrosing
alveolitis), identified histologically as VIP described above,
accounts for 50% of IIP cases. IPF is referred to as pneumonia, the
role of inflammation of IPF is insignificant, and environmental,
genetic, or other unknown factors are thought to initially trigger
alveolar epithelial cell injury. Self-perpetuating and aberrant
interstitial fibroblast and mesenchymal cell proliferation (with
collagen deposition and fibrosis) are thought to account for
development of clinical disease. Most patients have moderate to
advanced clinical disease at the time of diagnosis and the disease
deteriorates despite treatment. Currently, no specific treatment
method for IPF has been proven effective. Corticosteroids and
cytotoxic drugs (cyclophosphamide, azathioprine) have traditionally
been administered to IPF patients empirically in an attempt to halt
the progression of inflammation, but limited data support their
efficacy (Non Patent Document 1).
[0004] The beneficial effects of antifibrotic agents, pirfenidone
and nintedanib, have been confirmed using a general pulmonary
fibrosis model, "bleomycin model". However, the pathological
conditions of the "bleomycin model" do not always match those of
pulmonary fibrosis, and pirfenidone and nintedanib have been
confirmed to have only an effect of suppressing disease
progression. Furthermore, it has been reported that in clinical
trials using drugs suggested to have efficacy by the use of the
"bleomycin model", no effective therapeutic effect on IPF patients
has been obtained in most cases (Non Patent Document 2). Even if
effects on lung diseases have been confirmed with the use of the
bleomycin model, however, it cannot be currently said that the
effects can be actually confirmed on pulmonary fibrosis. Moreover,
lung transplantation is employed as a treatment method for
pulmonary fibrosis, but is problematic in lack of donors and
rejection upon transplantation. Accordingly, development of a novel
treatment method for pulmonary fibrosis has been desired.
[0005] Meanwhile, mesenchymal stein cells are pluripotent
progenitor cells that were isolated from the bone marrow for the
first time by Friedenstein (1982) (Non Patent Document 3). The
presence of the mesenchymal stem cells in various tissues including
hone marrow, umbilical cord, fat, and the like has been revealed.
Mesenchymal stem cell transplantation is expected as a novel
treatment method for various intractable diseases (Patent Documents
1 to 4). It is currently known that cells having functions
equivalent to those of mesenchymal stem cells are present among
mesenchymal stromal cells of adipose tissue arid fetal appendages
such as placenta, umbilical cord, and egg membrane. Hence, the
mesenchymal stein cells may also be referred to as mesenchymal
stromal cells.
PRIOR ART DOCUMENTS
Patent Documents
[0006] Patent Document 1: JP 2002-506831 A
[0007] Patent Document 2: JP 2000-508911 A
[0008] Patent Document 3: JP 2012-157263 A
[0009] Patent Document 4: JP 2012-508733 A
Non Patent Documents
[0010] Non Patent Document 1: The Merck Manual of Diagnosis and
Therapy, 18th Edition, Japanese version, pp. 466-471, Feb. 22,
2007, published by Nikkei B P
[0011] Non Patent Document 2: Timothy S. B. et al., American
Journal of Respiratory and Critical Care Medicine, p. 214-222,
189(2), 2014
[0012] Non Patent Document 3: Pittenger F. M. et al., Science 284,
pp. 143-147, 1999
SUMMARY OF INVENTION
Technical Problem
[0013] Under the above circumstances, an object of the present
invention is to provide a novel therapeutic agent for pulmonary
fibrosis, particularly idiopathic pulmonary fibrosis (IPF), for
which currently no effective pharmacotherapy has been
established.
Solution to Problem
[0014] In order to achieve the above object, the present inventors
verified therapeutic effects of mesenchymal stem cells on pulmonary
fibrosis using cells collected from tissues having lesions of
pulmonary fibrosis. As a result, the inventors found that
mesenchymal stem cells can inhibit fibrosis. The present invention
provides a novel therapeutic agent for pulmonary fibrosis. The gist
of the present invention is as follows. [0015] [1] A therapeutic
agent for pulmonary fibrosis comprising mesenchymal stein cells.
[0016] [2] The therapeutic agent for pulmonary fibrosis according
to [1], wherein pulmonary fibrosis is usual interstitial pneumonia
(UIP) or idiopathic pulmonary fibrosis (IPF). [0017] [3] The
therapeutic agent for pulmonary fibrosis according to [1] or [2],
wherein the mesenchymal stem cells are derived from an adipose
tissue. [0018] [4] The therapeutic agent for pulmonary fibrosis
according to any one of [1] to [3], wherein the mesenchymal stem
cells are allogenic, [0019] [5] A therapeutic agent for pulmonary
fibrosis comprising a PTPRR expression promotor. [0020] [6] The
therapeutic agent for pulmonary fibrosis according to [5], wherein
the PTPRR expression promotor dephosphorylates phospholated ERK.
[0021] [7] A kit for treatment of pulmonary fibrosis, comprising
the therapeutic agent for pulmonary fibrosis according to any one
of [1] to [6], a container, and a label.
Advantageous Effects of Invention
[0022] According to the present invention, a novel therapeutic
agent for pulmonary fibrosis, a PTPRR expression promotor and a
novel kit for treatment of pulmonary fibrosis can be provided.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 shows the effects of the therapeutic agent for
pulmonary fibrosis of the present invention on the fibrosis-related
gene expression (.alpha.-SMA, CDH2, Collagen 1) in cells derived
from lesions of pulmonary fibrosis.
[0024] FIG. 2 shows the effects of the therapeutic agent for
pulmonary fibrosis of the present invention on dephosphorylation of
phosphorylated ERK molecules within cells derived from lesions of
pulmonary fibrosis.
[0025] FIG. 3 shows the effects of the therapeutic agent for
pulmonary fibrosis of the present invention on fibrosis-related
gene expression (.alpha.-SMA, CDH2, FN1) in alveolar
epithelium-like cell lines.
[0026] FIG. 4 shows the histological images of the lungs of a
severe combined immunodeficient mouse (pulmonary fibrosis model) to
which cells derived from lesions of pulmonary fibrosis were
administered.
DESCRIPTION OF EMBODIMENTS
[0027] The therapeutic agent for pulmonary fibrosis and the kit for
treatment of pulmonary fibrosis of the present invention will be
described in detail.
<Therapeutic Agent for Pulmonary Fibrosis>
[0028] The therapeutic agent for pulmonary fibrosis of the present
invention comprises mesenchymal stem cells. Mesenchymal stem cells
act on the cells of involved tissues of fibrosing lungs, exerting
an effect of lowering the expression of a fibrosis-related gene by
cells. Moreover, administration of mesenchymal stem cells to
animals with fibrosis can be expected not only to be effective at
the level of cells, hut also to alleviate fibrosis. The therapeutic
agent for pulmonary fibrosis in the present invention may comprise,
in addition to mesenchymal stem cells, other components unless the
effects of the present invention are not deteriorated. Mesenchymal
stem cells and other components will be described in detail as
follows.
[0029] (Mesenchymal Stem Cells)
[0030] The term "mesenchymal stem cells" in the present invention
refers to cells being capable of differentiating into one or more
cells, preferably two or more cells, and further preferably three
or more cells belonging to the mesenchyme (bone cells,
cardiomyocytes, cartilage cells, tendon cells, fat cells and the
like), and capable of proliferating while keeping the capability.
The term "mesenchymal stem cells" as used in the present invention
refers to cells same as mesenchymal stromal cells and the two are
not particularly differentiated. In addition, the term is simply
denoted as mesenchymal cells. Examples of tissue containing
mesenchymal stem cells include adipose tissue, umbilical cord, bone
marrow, umbilical cord blood, endometrial, placenta, amnion,
chorion, decidua, dermis, skeletal muscle, periosteum, dental sac,
periodontal ligament, dental pulp, and tooth germ. For example, the
term "adipose tissue-derived mesenchymal stem cells" refers to
mesenchymal stem cells contained in adipose tissues, and may also
be referred to as adipose tissue-derived cnesenchycnal stromal
cells. Of these, in view of efficacy for treatment of pulmonary
fibrosis, the ease of availability, and the like, adipose
tissue-derived mesenchymal stem cells, umbilical cord-derived
mesenchymal stem cells, bone marrow-derived mesenchymal stem cells,
placenta-derived mesenchymal stem cells, and dental pulp-derived
mesenchymal stem cells are preferable, adipose tissue-derived
mesenchymal stem cells and umbilical cord-derived mesenchymal stem
cells are more preferable, and adipose tissue-derived mesenchymal
stem cells are even more preferable.
[0031] Mesenchymal stem cells in the present invention may be
derived from the same species as or different species from that of
a subject to be treated (test subject). Examples of the species of
mesenchymal stein cells in the present invention include human,
monkey, horse, cattle, sheep, pig, dog, cat, rabbit, mouse and rat,
and preferably the mesenchymal stem cells are cells derived from
the same species as that of a subject to be treated (test subject).
The mesenchymal stem cells in the present invention may be derived
from a subject to be treated (test subject); that is, isogenic
(autologous)cells, or may be derived from another subject of the
same species; that is, may be allogeneic cells. The mesenchymal
stem cells are preferably allogeneic cells.[0017] Mesenchymal stem
cells are unlikely to cause rejection also in an allogenic test
subject. Therefore, previously prepared donor cells subjected to
expansion culture and then cryopreservation can be used as
mesenchymal stem cells in the therapeutic agent for pulmonary
fibrosis of the present invention. Accordingly, compared with a
case in which autologous mesenchymal stem cells are prepared for
use, mesenchymal stem cells in the present invention are more
preferably allogenic in view of easy commercialization and ease of
obtaining some stable effectiveness.
[0032] The term "mesenchymal stem cells" in the present invention
refers to any cell population containing mesenchymal stem cells.
Such a cell population comprises at least 20% or more, preferably
30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or
99% of mesenchymal stem cells.
[0033] The term "adipose tissue" in the present invention refers to
tissue containing fat cells and mesenchymal stem cells including
microvascular cells and the like, which can be obtained through
surgical resection or suction of subcutaneous fat of mammals, for
example. Adipose tissue can be obtained from subcutaneous fat.
Adipose tissue is preferably obtained from animals of the same
species as that of a test subject to which adipose tissue-derived
mesenchymal stem cells are administered as described later. In view
of administration to humans, adipose tissue is more preferably
human subcutaneous fat. A donor (individual) from which
subcutaneous fat is supplied may be alive or dead, however, adipose
tissue to be used in the present invention is preferably tissue
collected from a living individual. When adipose tissue is
collected from an individual, examples of liposuction include PAL
(power-assisted) liposuction, elcornia laser liposuction, and body
jet liposuction. In view of maintaining cell status, preferably no
ultrasonic wave is used.
[0034] The umbilical cord in the present invention is a white
tubular tissue connecting between the fetus and the placenta, is
composed of umbilical cord veins, umbilical cord arteries, mucous
connective tissue (Wharton's Jelly), umbilical cord matrix itself,
and the like, and is rich in mesenchymal stein cells. The umbilical
cord is preferably obtained from animals of the same species as
that of a test subject (a subject to which the agent is
administered) for which the therapeutic agent for pulmonary
fibrosis of the present invention is used. In view of
administration of the therapeutic agent for pulmonary fibrosis of
the present invention to humans, the umbilical cord is more
preferably human umbilical cord.
[0035] The term "bone marrow" in the present invention refers to
spongy tissue filling the bone lumen and is a hematopoietic organ.
Bone marrow aspirate is present in the bone marrow, and cells
existing therein are referred to as "bone marrow cells". Bone
marrow cells include, in addition to erythrocytes, granulocytes,
megakaryocytes, lymphocytes, fat cells and the like, mesenchymal
stem cells, hematopoietic stem cells, vascular endothelial
precursor cells, and the like. Bone marrow cells can be collected
from human ilia, long bones, or other bones, for example.
[0036] The term "mesenchymal stem cells derived from each tissue"
in the present invention such as adipose tissue-derived mesenchymal
stem cells, umbilical cord-derived inesenchyinal stem cells, and
bone marrow-derived inesenchyinal stein cells refer to any cell
population containing mesenchymal stem cells derived from each
tissue such as adipose tissue-derived mesenchymal stem cells,
umbilical cord-derived mesenchymal stem cells, and bone
marrow-derived mesenchymal stern cells, respectively. Such a cell
population comprises at least 20% or more, preferably 30%, 40%,
50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% of
mesenchymal stem cells derived from each tissue such as adipose
tissue-derived mesenchymal stem cells, umbilical cord-derived
meserichymal stem cells, and bone marrow-derived mesenchymal stein
cells.
[0037] Mesenchymal stein cells in the present invention may be
characterized by growth characteristics (e.g, population doubling
capability or doubling time required from passages to senescence),
karyotype analysis (e.g., normal karyotype; maternal or neonatal
sequence), surface marker expression as determined by flow
cytometry (e.g, FACS analysis), immunohistochemistry and/or
immunocytochemistry (e.g, epitope detection), gene expression
profiling (e.g., gene chip arrays; polymerase chain reaction such
as reverse transcription PCR, real time PCR, and conventional
PCR)), miRNA expression profiling, protein arrays, secretion of
proteins such as cytokines (e.g., analysis of plasma clotting,
ELISA, and cytokine arrays) metabolites metabolome analysis), other
methods known in the art, and the like.
[0038] (Method for Preparing Mesenchymal Stem Cells)
[0039] Mesenchymal stem cells can be prepared by a method
well-known by persons skilled in the art. A method for preparing
adipose tissue-derived mesenchymal stem cells is described below as
an example. Adipose tissue-derived mesenchymal stem cells may be
obtained, for example, by the production method disclosed in U.S.
Pat. No. 6,777,231, and can be produced, for example, by a method
comprising the following steps (i) to (iii): [0040] (i) step of
obtaining a cell suspension by enzymatic digestion of adipose
tissue; [0041] ( step of precipitating cells for re-suspension of
cells in an appropriate medium; and [0042] (iii) step of culturing
cells on a solid surface and then removing cells not binding to the
solid surface.
[0043] Adipose tissue washed in advance is preferably used in step
(i). Washing can be performed using a physiologically compatible
saline solution (e.g., phosphate-buffered saline (PBS)) and
vigorously stirring for precipitation. This is performed for
removing contaminants (also referred to as debris such as damaged
tissue, blood, and erythrocytes) contained in adipose tissue from
the tissue. Therefore, washing and precipitation are generally
repeated until debris is removed overall from the supernatant.
Remaining cells are present as various sized masses and are
dissociated with minimum damage to the cells themselves. Hence,
cell mass after washing is preferably treated with an enzyme (e.g.,
collagenase, dispase or trypsin) that weakens or disrupts
cell-to-cell binding. The amount of and the period for treatment
with such an enzyme are varied depending on conditions to be
employed, but are well-known in the art. Instead of or in
combination with such enzymatic treatment, cell mass can be
dissociated by other treatment methods such as mechanical stirring
or a method using ultrasonic energy, thermal energy or the like. In
order to minimize damage to cells, enzymatic treatment alone is
preferably employed. When an enzyme is used, it is desired to
inactivate the enzyme using medium and the like after an
appropriate period of time in order to minimize harmful effects on
cells.
[0044] A cell suspension obtained in step (i) contains aggregated
cell slurry or suspension, and various contaminating cells such as
erythrocytes, smooth muscle cells, endothelial cells, and
fibroblasts. Therefore, aggregated cells and these contaminating
cells may be subsequently separated and removed. However, since
these cells can be removed by adhesion and washing in step (iii)
described later, such separation and removal can be omitted. When
contaminating cells are separated and removed, separation and
removal can he achieved by centrifugation that forcibly separates
cells into a supernatant and a precipitate. The precipitate thus
obtained containing contaminating cells is suspended in a
physiologically compatible solvent. The suspended cells may include
erythrocytes. However, the erythrocytes are removed by selection
that involves adhesion thereof to the solid surface described
later, a step of lysis is not always required. As a method for
selectively lysing erythrocytes, for example, a method well-known
in the art such as incubation in hypertonic medium or hypotonic
medium using lysis with ammonium chloride can be employed. After
lysis, the lysate may be separated from desired cells, for example,
by filtration, centrifugal sedimentation, fractionation by density,
or the like.
[0045] In step (ii), suspended cells may be washed once or multiple
times successively, centrifuged, and then re-suspended in medium in
order to increase the purity of mesenchymal stem cells. In addition
to these treatments, cells may be separated on the basis of cell
surface marker profile or cell size and the granular nature of the
cells.
[0046] A medium to be used for re-suspension is not particularly
limited as long as it is a medium in which mesenchymal stem cells
can be cultured. Such a medium may also be prepared by adding
serum, and/or one or more serum substitutes such as albumin,
transferrin, fatty acid, insulin, sodium selenite, cholesterol,
collagen precursor, trace element, 2-mercaptoethanol, and
3'-thiolglycerol to a basal medium. These media may be further
supplemented with substances such as lipids, amino acids, proteins,
polysaccharides, vitamins, growth factors, low-molecular-weight
compounds, antibiotics, antioxidants, pyruvic acids, buffer, and
inorganic salts, as necessary.
[0047] Examples of the above basal medium include IMDM, Medium 199,
Eagle's Minimum Essential Medium (EMEM), .alpha.MEM, Dulbecco's
Modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium,
Fischer's medium, MCDB201 medium and mixed media thereof,
[0048] Examples of the above serum include, but are not limited to,
human serum, fetal bovine serum (FBS), bovine serum, fetal calf
serum, goat serum, horse serum, porcine serum, sheep serum, rabbit
serum, and rat serum. When such serum is used, it may be added in
an amount of 5 v/v % to 15 v/v %, preferably 10 v/v % relative to
the basal medium.
[0049] Examples of the above fatty acid include, but are not
limited to, linoleic acid, oleic acid, linolenic acid, arachidonic
acid, myristic acid, palmitoyl acid, palmitic acid, and stearic
acid. Examples of lipids include, but are not limited to,
phosphatidyl serine, phosphatidyl ethanolamine, and phosphatidyl
choline. Examples of amino acids include, but are not limited to.
L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine,
L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of
proteins include, but are not limited to, ecotin, reduced
glutathione, fibronectin and .beta.2-microglobulin. Examples of
polysaccharides include, but are not limited to, glycosaminoglycans
and, of these glycosaminoglycans, particularly hyaluronic acid, and
heparan sulfate. Examples of growth factors include, but are not
limited to, platelet-derived growth factor (PDGF), basic fibroblast
growth factor (bFGF), transforming growth factor beta (TGF-.beta.),
hepatocyte growth factor (HGF), epidermal growth factor (EGF),
connective tissue growth factor (CTGF), and vascular endothelial
cell growth factor (VEGF). From the view point of using
adipose-derived mesenchymal stem cells obtained in the present
invention for cell transplantation, it is preferable to use a
(Xeno-Free) medium containing no heterologous components such as
serum. As a medium, a commercially available ready-made medium for
mesenchymal stem cells (stromal cells) is provided by a
manufacturer such as PromoCell GmbH, Lonza, Biological Industries,
Veritas, R&D Systems, Corning Inc. and Rohto Pharmaceutical
Co., Ltd.
[0050] Subsequently, in step (iii), cells are cultured on a solid
surface without allowing differentiation of cells in the cell
suspension obtained in step (ii), using any appropriate cell medium
as described above, appropriate cell density and culture
conditions. In the present invention, the term "solid surface"
refers to any material that enables binding and adhesion of adipose
tissue-derived mesenchymal stem cells in the present invention. In
a specific aspect, such a material is a plastic material treated to
promote binding and adhesion of mammalian cells onto the surface.
The shape of a culture vessel having a solid surface is not
particularly limited, and petri dishes, flasks, and the like are
used suitably. To remove unbound cells and cell fragments, cells
are washed after incubation.
[0051] In the present invention, finally cells remaining bound and
adhered onto the solid surface can be selected as cell populations
of adipose tissue-derived mesenchymal stem cells.
[0052] To confirm that the cells thus selected are adipose
tissue-derived mesenchymal stem cells in the present invention, the
cells may be analyzed for surface antigens by a conventional method
using flow cytometry or the like. Moreover, the cells may also be
tested for their ability to differentiate into each cell lineage,
and such differentiation can be performed by a conventional
method.
[0053] Mesenchymal stem cells in the present invention can be
prepared as described above, and may be defined as cells having the
following properties of: [0054] (1) exhibiting adhesion to plastic
under culture conditions of standard medium; [0055] (2) being
positive for surface antigens CD44, CD73, and CD90, and negative
for CD31 and CD45; and [0056] (3) being capable of differentiating
into bone cells, fat cells, and cartilage cells under culture
conditions.
[0057] (Cryopreservation of Mesenchymal Stem Cells)
[0058] Mesenchymal stem cells in the present invention may be cells
repeatedly cryopreserved and thawed as appropriate, as long as the
cells have therapeutic effects on pulmonary fibrosis. In the
present invention, cryopreservation can be performed by suspending
mesenchymal stern cells in a cryopreservation solution well-known
by persons skilled in the art and then cooling the resultant.
Suspension can be performed by dissociating cells using a
cell-dissociating agent such as trypsin, transferring cells into a
cryopreservation container, treating as appropriate, and then
adding a cryopreservation solution.
[0059] A cryopreservation solution may contain DMSO (Dimethyl
sulfoxide) as a cryoprotectant. However, DMSO has a property of
inducing differentiation of mesenchymal stern cells in addition to
cytotoxicity, and thus reducing the DMSO content is preferred.
Examples of an alternative to DMSO include glycerol, propylene
glycol or polysaccharides, and sugar alcohols. When DMSO is used,
the concentration of DMSO in a cryopreservation solution ranges
from 5% to 20%, preferably 5% to 10%, and is more preferably 10%.
In addition to this, the cryopreservation solution may also contain
an additive disclosed in WO2007/058308. As such a cryopresmation
solution, for example, a cryopreservation solution provided from
BioVerde, NIPPON Genetics, ReproCELL, ZENOAQ, COSMO BIO, Kohjin Bio
Co., Ltd.. Thermo Fisher Scientific K.K. or the like may be
used.
[0060] When the above-suspended cells are cryopreserved, the cells
may be stored at a temperature between -80.degree. C. and
-100.degree. C. (e.g., -80.degree. C.) using any freezer that can
reach the temperatures. The type of a freezer is not particularly
limited, and a program freezer may be used to control the cooling
rate as appropriate in order to avoid any rapid temperature change.
The cooling rate may be selected as appropriate depending on the
components of a cryopreservation solution and cooling can be
performed according to the instructions of the manufacturer of the
cryopreservation solution.
[0061] The upper limit of the preservation period is not
particularly specified, as long as cells retain their
characteristics equivalent to those before freezing, after thawing
of the cryopreserved cells under the above conditions. Examples of
the preservation period include 1 week or more, 2 weeks or more, 3
weeks or more, 4weeks or more, 2 months or more, 3 months or more,
4 months or more, 5 months or more, 6 months or more, 1 year or
more, or periods longer than these. Cell damage can be inhibited by
preserving cells at a lower temperature, and thus cells may be
transferred to and then preserved in a gas phase over liquid
nitrogen (about -150.degree. C. or lower and -180.degree. C. or
higher). When cells are preserved in a gas phase over liquid
nitrogen, preservation can be performed using a preservation
container well-known by persons skilled in the art. Cells are
preferably preserved in a gas phase over liquid nitrogen when the
cells are preserved, for example, for 2 weeks or more, and a way of
preservation is not particularly limited thereto.
[0062] Thawed mesenchymal stem cells may be cultured as appropriate
until the next cryopreservation. Mesenchymal stem cells are
cultured using a medium in which the above mesenchymal stein cells
can be cultured without particular limitation and may also be
cultured at culture temperatures ranging from about 30.degree. C.
to 40.degree. C., and at preferably about 37.degree. C. under an
atmosphere of CO.sub.2-containing air. CO.sub.2 concentration
ranges from about 2% to 5%, and is preferably about 5%. Upon
culturing, after the culture reaches appropriate confluency for the
culture vessel (for example, 50% to 80% of the culture vessel is
occupied by cells), cells may be dissociated using a dissociating
agent such as trypsin, and then seeded at appropriate cell density
for a separately prepared culture vessel, and may be continuously
cultured. Upon seeding of cells, examples of typical cell density
include 100 cells/cm.sup.2 to 100,000 cells/cm.sup.2, 500
cells/cm.sup.2 to 50,000 cells/cm.sup.2, 1,000 to 10,000
cells/cm.sup.2, and 2,000 to 10,000 cells/cm.sup.2. In a specific
aspect, cell density ranges from 2,000 to 10,000 cells/cm.sup.2.
The period required for the culture to reach appropriate confluency
is preferably adjusted to range from 3 days to 7 days. During
culture, medium exchange may be, as appropriate, performed as
necessary.
[0063] Cryopreserved cells can be thawed by a method well-known by
persons skilled in the art. For example, a method is exemplified,
in which cryopreserved cells are thawed by letting the cells stand
or shaking the cells within a thermostatic bath or a water bath at
room temperature to 37.degree. C. and preferably 37.degree. C.
[0064] (Form of Mesenchymal Stem Cells)
[0065] The mesenchymal stem cells of the present invention may be
cells in any form and examples thereof include cells recovered
after dissociation of cells in the culture or cells frozen in a
cryopreservation solution. The use of cells prepared by subdividing
cells of the same lot obtained via expansion culture into small
quantities followed by cryopreservation is preferable in terms of
stably obtaining similar beneficial effects, excellent
handleability, and the like. Mesenchymal stem cells in a
cryopreserved form are thawed immediately before use, and then
directly mixed with a solution for suspension of mesenchymal stem
cells, such as an infusion solution or a medium while being
suspended in a cryopreservation solution. Furthermore, a
cryopreservation solution is removed by a method such as
centrifugation and then the cells may be suspended in a solution
for suspension of mesenchymal stem cells, such as an infusion
solution or a medium. Here, the term "infusion solution" in the
present invention refers to a solution that is used for treating
humans. Examples thereof include, but are not particularly limited
to, a saline solution, the Japanese Pharmacopoeia physiological
saline, 5% glucose solution, the Japanese Pharmacopoeia glucose
injection, Ringer's solution, the Japanese Pharmacopoeia Ringer's
solution, Ringer's lactate, Ringer's acetate, No. 1 fluid (starter
solution), No. 2 fluid (fluid replacement for dehydration), No. 3
fluid (maintenance fluid), and No. 4 fluid (postoperative recovery
fluid). Note that the above solutions for suspension of mesenchymal
stem cells, such as infusion solutions or media may be prepared to
contain other components (pharmaceutically acceptable carriers and
additives) described later.
[0066] The therapeutic agent for pulmonary fibrosis of the present
invention may comprise, in addition to the above mesenchymal stem
cells, pharmaceutically acceptable carriers and additives according
to the application and the form thereof and a standard method, as
long as they do not deteriorate the effects of the present
invention. Examples of such carriers and additives include, but are
not limited to, tonicity adjusting agents, thickeners, saccharides,
sugar alcohols, preservatives, antiseptics or antimicrobial agents,
pH adjusters, stabilizers, chelating agents, oily bases, gel bases,
surfactants, suspending agents, binders, excipients, lubricants,
disintegrants, foaming agents, fluidizing agents, dispersants,
emulsifiers, buffering agents, solubilizing agents, antioxidants,
sweetening agents, acidulants, colorants, flavoring agents, and
aroma chemicals or refreshing agents. Examples of typical
components include the following carriers and additives.
[0067] Examples of carriers include aqueous carriers such as water
and hydrous ethanol. Furthermore, examples of tonicity adjusting
agents (inorganic salts) include sodium chloride, potassium
chloride, calcium chloride, and magnesium chloride. Examples of
polyhydric alcohols include glycerin, propylene glycol, and
polyethylene glycol. Examples of thickeners include carboxyvinyl
polymer, hydroxyethylcellulose, hydroxypropyl methylcellulose,
methylcellulose, alginic acid, polyvinyl alcohol (complete or
partially saponified product), polyvinyl pyrrolidone, and macrogol.
Examples of saccharides include cyclodextrin and dextrose. Examples
of sugar alcohols include xylitol, sorbitol, and mannitol (may be
d-, l- or dl-isomer). Examples of preservatives, antiseptics or
antimicrobial agents include dibutylhydroxytoluen, butylated
hydroxyanisole, alkyldiamino ethylglycine hydrochloride, sodium
benzoate, ethanol, benzalkonium chloride, benzethonium chloride,
chlorhexidine gluconate, chlorobutanol, sorhic acid, potassium
sorbate, trometamol, sodium dehydroacetate, methyl
parahydroxybenzoate, ethyl parahydroxybenzoate, propyl
parahydroxybenzoate, butyl parahydroxybenzoate, oxyquinoline
sulfate, phenethyl alcohol, benzyl alcohol, higuanide compound
(specific examples thereof include polihexanide hydrochloride
(polyhexamethylene biguanide) and the like), and Grokiru (brand
name, manufactured by Rhodia Corporation). Examples of pH adjusters
include hydrochloric acid, boric acid, aminoethanesulfonic acid,
epsilon-aminocaproic acid, citric acid, acetic acid, sodium
hydroxide, potassium hydroxide, calcium hydroxide, magnesium
hydroxide, sodium hydrogencarbonate, sodium carbonate, borax,
triethanolamine, monoethanolamine, diisopropanolamine, sulfuric
acid, magnesium sulfate, phosphoric acid, polyphosphoric acid,
propionic acid, oxalic acid, gluconic acid, fumaric acid, lactic
acid, tartaric acid, malic acid, succinic acid, gluconolactone, and
ammonium acetate. Examples of stabilizers include
dibutylhydroxytoluen, trometamol, sodium formaldehydosulfoxylate
(rongalit), tocopherol, sodium pyrosulfite, monoethanolamine,
aluminum monostearate, glyceryl monostearate, sodium hydrogen
sulfite, and sodium sulfite. Examples of oily bases include
vegetable oils such as olive oil, corn oil, soybean oil, sesame
oil, and cotton seed oil and medium-chain triglyceride. Examples of
aqueous bases include Macrogol 400. Examples of gel bases include
carboxyvinyl polymer and gum substance. Examples of surfactants
include polysorbate 80, hardened castor oil, glycerin fatty acid
ester, and sorbitan sesquioleate. Examples of suspending agents
include white beeswax and various surfactants, gum arabic, gum
arabic powder, xanthan gum, and soybean lecithin. Examples of
binders include hydmxyethylcellulose, hydroxypmpylcellulose,
hydroxypropyl methylcellulose, sodium carboxymethylcellulose,
polyvinyl pyrrolidone, and polyvinyl alcohol. Examples of
excipients include sucrose, lactose, starch, corn starch,
microcrystalline cellulose, and light silicic anhydride. Examples
of lubricants include sucrose fatty acid ester, magnesium stearate,
and talc. Examples of disintegrants include low substituted
hydroxypropylcellulose, crospovidone, and croscarmellose sodium.
Examples of foaming agents include sodium hydrogencarbonate.
Examples of fluidizing agents include sodium metasilicate aluminate
and light silicic anhydride.
[0068] The therapeutic agent for pulmonary fibrosis of the present
invention can be provided in various forms according to purposes,
such as various dosage forms including solids, semi-solids, liquids
and the like. Examples of dosage forms that can be used include
solids (e.g., tablets, powders, powdered drugs, granules and
capsules), semi-solids [ointments (e.g., hard ointment and soft
ointment), cream pharmaceuticals and the like], liquids [e.g.,
lotions, extract agents, suspensions, emulsions, syrups, injection
preparations (including infusion solutions, embedded injection
preparations, persistent injections, and injection preparations
prepared before use), dialysis agents, aerosol agents, soft
capsules, and drinkable preparations], adhesive preparations, and
adhesive skin patches. Moreover, the therapeutic agent for
pulmonary fibrosis of the present invention can also be used in the
forms of solution, milky lotion, or the like in oily or aqueous
vehicles. Further, the therapeutic agent for pulmonary fibrosis of
the present invention can also be applied to affected parts via
spraying. The therapeutic agent for pulmonary fibrosis of the
present invention can also be gelatinized or formed into a sheet
and then used after spraying to an affected part. The therapeutic
agent for pulmonary fibrosis of the present invention can be
applied to an affected part after the above mesenchymal stem cells
are formed into a sheet or a tertiary structure.
[0069] The therapeutic agent for pulmonary fibrosis of the present
invention can be used by suspending or diluting the above-described
mesenchymal stem cells and other components (pharmaceutically
acceptable carriers and additives) in/with an infusion solution
such as a saline solution, the Japanese Pharmacopoeia physiological
saline, 5% glucose solution, the Japanese Pharmacopoeia glucose
injection, Ringer's solution, the Japanese Pharmacopoeia Ringer's
solution, Ringer's lactate, Ringer's acetate, Ringer's bicarbonate,
No. 1 fluid (starter solution), No. 2 fluid (fluid replacement for
dehydration), No. 3 fluid (maintenance fluid), No. 4 fluid
(postoperative recovery fluid), or the like, or, a solution for
suspension of mesenchymal stem cells such as cell culture medium,
e.g., DMEM. Preferably, the therapeutic agent for pulmonary
fibrosis can be used after suspension or dilution with
physiological saline, 5% glucose solution, No. 1 fluid (starter
solution), and more preferably 5% glucose solution and No. 1 fluid
(starter solution). Moreover, the solution for suspension of
mesenchymal stem cells may be prepared in such a manner that the
solution contains in advance the above other components
(pharmaceutically acceptable carriers and additives).
[0070] The therapeutic agent for pulmonary fibrosis of the present
invention may also be used in such a manner that mesenchymal stem
cells and a solution for suspension of mesenchymal stem cells are
separately enclosed and stored in different containers and then the
two may be mixed before use. Note that upon storage, the above
mesenchymal stem cells and solution for suspension of mesenchymal
stern cells may be in a frozen form or a refrigerated form.
[0071] When the therapeutic agent for pulmonary fibrosis of the
present invention is liquid, the pH of the therapeutic agent for
pulmonary fibrosis is not particularly limited as long as the pH is
within pharmaceutically, pharmacologically (pharmaceutically) or
physiologically acceptable range, and examples of such range
include a pH range of 2.5 to 9.0, preferably a pH range of 3.0 to
8.5, and more preferably a pH range of 3.5 to 8.0. Note that when
mesenchymal stem cells and a solution for suspension of mesenchymal
stem cells are separately enclosed and stored in different
containers, only the solution for suspension of mesenchymal stem
cells is required to meet the above conditions,
[0072] When the therapeutic agent for pulmonary fibrosis of the
present invention is liquid, the osmotic pressure of the
therapeutic agent for pulmonary fibrosis is not particularly
limited, as long as the osmotic pressure is within the range
acceptable by a live body. Examples of the osmotic pressure ratio
of the therapeutic agent for pulmonary fibrosis of the present
invention include the ratios ranging from preferably 0.7 to 5.0,
more preferably 0.8 to 3.0, and further preferably 0.9 to 1.4. The
adjustment of the osmotic pressure can be carried out by a
well-known method in the art with an inorganic salt, a polyhydric
alcohol, a sugar alcohol, a saccharide, or the like. The osmotic
pressure ratio is defined as a ratio of an osmotic pressure of a
sample to 286 mOsm (osmotic pressure of an aqueous 0.9 w/v % sodium
chloride solution) as prescribed in The Japanese Pharmacopeia
Fifteenth Edition, and an osmotic pressure is measured referring to
a method for measuring an osmotic pressure (cryoscopic method) as
prescribed in The Japanese Pharmacopeia. Here, the standard
solution for measuring an osmotic pressure ratio (aqueous 0.9 w/v %
sodium chloride solution) is prepared by drying sodium chloride
(The Japanese Pharmacopeia standard reagent) at a temperature
between 500.degree. C. and 650.degree. C. for 40 to 50 minutes, and
thereafter allowing it to cool in a desiccator (silica gel),
accurately weighing 0.900 g of the cooled sample, and dissolving
the sample in purified water to precisely make up a volume of 100
or alternatively, a commercially available standard solution for
measuring an osmotic pressure ratio (aqueous 0.9 w/v % sodium
chloride solution) is used. Note that if mesenchymal stem cells and
a solution for suspension of mesenchymal stem cells are separately
enclosed and stored in different containers, only the solution for
suspension of mesenchymal stem cells is required to meet the above
conditions.
[0073] Examples of the route of administration of the therapeutic
agent for pulmonary fibrosis of the present invention to a subject
include peroral administration, subcutaneous administration,
intramuscular administration, intravenous administration,
intraarterial administration, intrathecal administration,
intraperitoneal administration, sublingual administration,
transrectal administration, transvaginal administration, transnasal
administration, inhalation, transdermal administration, implant,
and direct administration via. spraying over or applying a sheet or
the like onto the pulmonary surface. In view of the efficacy of the
therapeutic agent for pulmonary fibrosis of the present invention,
the routes of administration are preferably implant, intraarterial
administration, intravenous administration and direct
administration via spraying over or applying a sheet or the like
over the pulmonary surface. In view of lowering the burden on a
subject, the route of administration is more preferably
intra-pulmonary arterial administration or intravenous
administration, and is most preferably intravenous
administration.
[0074] The dose (dosage) of the therapeutic agent for pulmonary
fibrosis of the present invention can differ depending on a
patient's conditions (e.g., body weight, age, symptoms, and
physical condition), dosage form of the therapeutic agent for
pulmonary fibrosis of the present invention, and the like. In view
of having sufficient therapeutic effects on pulmonary fibrosis, a
higher dose tends to be preferred. On the other hand, in view of
suppressing the expression of adverse reaction, a lower dose tends
to be preferred. In general, when the agent is administered to an
adult, the number of cells ranges from 1.times.10.sup.3 to
1.times.10.sup.12 cells/administration, preferably 1.times.10.sup.4
to 1.times.10.sup.11 cells/administration, more preferably
1.times.10.sup.5 to 1.times.10.sup.10cells/administration, and
further preferably 5.times.10.sup.6 to 1.times.10.sup.9
cells/administration. Furthermore, the dosage per kg body weight of
a patient ranges from 1.times.10 to 5.times.10.sup.10 cells/kg,
preferably 1.times.10.sup.2 to 5.times.10.sup.9 cells/kg, more
preferably 1.times.10.sup.3 to 5.times.10.sup.8 cells/kg, and
further preferably 1.times.10.sup.4 to 5.times.10.sup.7 cells/kg.
Note that this amount of the agent may be considered as a single
dose and administered multiple times, or this amount of the agent
may be administered in divided doses.
[0075] The rate of administration of the therapeutic agent for
pulmonary fibrosis of the present invention to a subject can differ
depending on a patient's conditions (e.g., body weight, age,
symptoms, and physical condition), the route of administration of
the therapeutic agent for pulmonary fibrosis of the present
invention, and the like. In general, when the agent is administered
to an adult, the rate of administration ranges from 50 mL/h to
1,000 mL/h, preferably 75 mL/h to 500 mL/h, and more preferably 100
mL/h to 250 mL/h.
[0076] The temperature for administration of the therapeutic agent
for pulmonary fibrosis of the present invention to a subject can
differ depending on a patient's conditions (e.g., body weight, age,
symptoms, and physical condition), the route of administration of
the therapeutic agent for pulmonary fibrosis of the present
invention, and the like. In general, the temperature for
administration ranges from 4.degree. C. to 45.degree. C.,
preferably 15.degree. C. to 37.degree. C., and more preferably room
temperature to 37.degree. C.
[0077] The therapeutic agent for pulmonary fibrosis of the present
invention may also be administered with 1 or 2 or more other drugs.
Such a drug may be any drug that can be used as a drug for a
respiratory organ, and an immunosuppressive drug. Examples thereof
include dimorpholamine, doxapram hydrochloride hydrate, sivelestat
sodium hydrate, pulmonary surfactant, dornase alfa, pirfenidone,
interferon-.gamma.-1b, predonisone, corticosteroids, pirfenidone
(Pirespa (registered trademark)), nintedanib (Ofev (registered
trademark)), angiotensin receptor blocker, cyclosporin,
azathioprine, mizoribine, basiliximab, tacrolimus hydrate,
gusperimus hydrochloride, mycophenolate mofetil, everolimus, and
acetylcysteine. In addition, examples of a case in which the
therapeutic agent for pulmonary fibrosis of the present invention
is administered with 1 or 2 or more other drugs include various
cases such as a case in which the therapeutic agent for pulmonary
fibrosis of the present invention and other drugs are used
simultaneously, a case in which either one of them is administered
and then, after a lapse of a certain period of time, the other drug
is administered, and a combination thereof.
[0078] The therapeutic agent for pulmonary fibrosis of the present
invention can also be expressed as containing a PTPRR expression
promotor. Here, PTPRR is protein tyrosine phosphatase receptor-type
R, which is a type of receptor-type tyrosine phosphatase. PTPRR is
an enzyme that dephosphorylates tyrosine of a protein phosphorated
by tyrosine kinase. Receptor-type tyrosine phosphatase is composed
of an intracellular region having enzyme activity, a transmembrane
domain, and an extracellular region. A ligand binds to the
extracellular region so as to control the enzyme activity. The term
"PTPRR expression promotor" in the present invention is referred to
as a composition exhibiting an effect of promoting the expression
of PTPRR gene and/or PTPRR protein in cells or tissues. The PTPRR
expression promotor of the present invention is described as
follows.
[0079] [PTPRR Expression Promotor]
[0080] The PTPRR expression promotor in the present invention
promotes PTPRR expression in cells or tissues, which causes
pulmonary fibrosis, so as to enhance the dephosphorylation of
tyrosine-phosphorylated proteins. As a result, the PTPRR expression
promotor can suppress the cell proliferation and suppress tissue
fibrosis. Examples of such tyrosine-phosphorylated proteins include
ERK, JNK, and p38, and of these, ERK is preferable in view of
having an excellent effect of suppressing pulmonary fibrosis,
[0081] The PTPRR expression promotor of the present invention
preferably comprises mesenchymal stem cells having a function of
acting on cells or tissues that cause pulmonary fibrosis, so as to
promote the PTPRR expression. Description of mesenchymal stem cells
contained in the above therapeutic agent for pulmonary fibrosis can
be directly applied for mesenchymal stem cells contained in the
PTPRR expression promotor of the present invention.
[0082] Examples of diseases against which the therapeutic agent for
pulmonary fibrosis of the present invention is effective include
usual interstitial pneumonia (UIP), idiopathic pulmonary fibrosis
(IPF, idiopathic fibrosing alveolitis), nonspecific interstitial
pneumonia, bronchiolitis obliterans with organizing pneumonia
(organizing pneumonia of unknown etiology), interstitial lung
disease (ILD) with respiratory bronchiolitis, desquamative
interstitial pneumonia, acute interstitial pneumonitis (rapidly
progressive form of interstitial pneumonia, Hamman-Rich syndrome),
idiopathic interstitial pneumonia, drug-induced lung disease,
eosinophilic lung disease and chronic eosinophilic pneumonia. Of
these, the therapeutic agent for pulmonary fibrosis exhibits a
significant effect on usual interstitial pneumonia (UIP), and
idiopathic pulmonary fibrosis (IPF, idiopathic fibrosing
alveolitis).
[0083] <Kit for Treatment of Pulmonary Fibrosis t>
[0084] The present invention encompasses a kit for treatment of
pulmonary fibrosis, comprising mesenchymal stem cells and a
solution for suspension of mesenchymal stem cells. Description
given in the section of the therapeutic agent for pulmonary
fibrosis can be applied for mesenchymal stem cells and a solution
for suspension of mesenchymal stem cells contained in the kit of
the present invention.
[0085] Moreover, the kit for treatment of pulmonary fibrosis of the
present invention can be expressed as comprising the therapeutic
agent for pulmonary fibrosis of the present invention, a container
and a label. Examples of an appropriate container contained in the
kit of the present invention include, and are not particularly
limited to, cryotubes for freezing cells, bottles for solutions for
suspension of inesenchyinal stem cells, vials, and test tubes.
These containers may also be formed of various materials such as
glass, metal, plastic, or combinations thereof. The contents,
specifically mesenchymal stem cells, solutions for suspension of
mesenchymal stem cells, and the like, are described on the labels
of these containers.
[0086] The kit of the present invention can comprise other
materials desirable from commercial and users' viewpoints, such as
other additives, other drugs, diluents, filters, needles, syringes,
and package inserts containing directions for use.
[0087] <Treatment Method for Pulmonary Fibrosis and Method for
Promoting PTPRR Expression>
[0088] The present invention also encompasses a treatment method
for pulmonary fibrosis, wherein mesenchymal stem cells are used.
According to the treatment method of the present invention,
mesenchymal stem cells suppress the gene expression that promotes
pulmonary fibrosis, so that pulmonary fibrosis can be effectively
treated. Furthermore, the present invention encompasses a method
for promoting PTPRR expression in cells, tissues and the like,
wherein mesenchymal stem cells are used. According to the promoting
method of the present invention, mesenchymal stem cells act on
cells and tissues, so as to be able to promote PTPRR expression
effectively. Note that description given in the section of the
therapeutic agent for pulmonary fibrosis can be applied to describe
mesenchymal stein cells.
EXAMPLES
[0089] The present invention will be described specifically as
follows with reference to Examples and Test Examples, however, the
present invention is not limited by these Examples and the
like.
1. Preparation of Adipose Tissue-Derived Mesenchymal Stem Cells
(Example 1)
[0090] A serum free medium (Rohto) for mesenchymal stem cells was
added to commercially available cells (manufactured by Lonza,
PT-5006), and then the cell suspension was centrifuged at 400 g for
5 minutes. After removal of the supernatant, the resultant was
resuspended in a serum free medium for mesenchymal stein cells
(Rohto), and then cells were seeded in a flask. Cells were
subcultured at 37.degree. C. for several days in 5% CO.sub.2. After
few days, the culture product was washed with PBS to remove
remaining blood cells and adipose tissue contained in the culture
solution, thereby obtaining mesenchymal stem cells adhering to the
plastic container.
[0091] The adipose tissue-derived mesenchymal stem cells thus
obtained were aliquoted into centrifugation tubes, and then
centrifuged at 800 g for 5 minutes, so that cell precipitates were
obtained. After removal of the supernatants, an appropriate amount
of a cryopreserved cell solution (STEM-CELLBANKER (ZENOAQ)) was
added for suspension. The cell suspensions were aliquoted into
cryotubes, preserved within a freezer at -80.degree. C.,
transferred into gas phase over liquid nitrogen, and continuously
preserved. Hereinafter, adipose tissue-derived mesenchymal stem
cells are also referred to as ADMSC or ASC.
2. Effects of Adipose Tissue-Derived Mesenchymal Stem Cells on
Fibrosis-Related Gene Expression in UIP Patient-Derived Pulmonary
Fibroblasts (Example 2)
[0092] A specimen was collected from a patient diagnosed as having
lesions of pulmonary fibrosis and then operated due to lung cancer.
Sites (fibrosis (-)) observed to have no fibrosis and sites
(fibrosis (+)) observed to have fibrosis were distinguished from
each other, tissues of these sites were each cut into pieces,
culture thereof was started in 15% serum-containing DMEM
(manufactured by Wako) at 37.degree. C. In 10% CO.sub.2. On each of
days 1, 2, 4, and 7, medium exchange was performed after washing
with PBS (manufactured by Wako). Culture was performed for 10 days
in total, thereby obtaining UIP (usual interstitial pneumonia)
patient-derived pulmonary fibroblasts (hereinafter, referred to as
"UIP cells").
[0093] UIP cells (5.0.times.10.sup.4cells/well) suspended in 15%
serum-containing DMEM were seeded in each well of a 24-well plate,
and then cultured overnight within a CO.sub.2 incubator (37.degree.
C., 10% CO.sub.2). Each culture solution was exchanged with 1%
serum-containing DMEM. The above subcultured and cryopreserved
human adipose tissue-derived mesenchymal stem cells (Example 1)
were immersed in a thermostatic bath at 37.degree. C. to thaw the
cell suspension, and then collected in a 15 mL centrifugation tube.
The tube was filled with DMEM (manufactured by Wako) such a manner
that the volume reached 10 mL, and then centrifugation was
performed at room temperature and 400.times.g for 5 minutes
(manufactured by Eppendorf, 5702). Transwell inserts (Corning,
#3460) were set in a 24-well plate, to which 1% serum-containing
DMEM alone (Control), 1% serum-containing DMEM containing HGF with
a final concentration of 100 ng/ml, and human adipose
tissue-derived mesenchymal stem cells
(2.0.times.10.sup.5cells/well) suspended in 1% serum-containing
DMEM were added. Twenty four (24) hours after the start of
co-culture, RNA of UIP cells was collected, the mRNA expression
levels of fibrosis-related factors, ACTA2 (.alpha.-Smooth muscle
actin; .alpha.-SMA), CDH2 (Cadherin 2) and Collagen 1, were
measured by quantitative PCR. Furthermore, Western blot was
performed for UIP cells. The results of quantitative PCR are shown
in FIG. 1. Moreover, the results of Western blot are shown in FIG.
2.
[0094] As shown in FIG. 1, co-culture of UIP cells and human
adipose tissue-derived mesenchymal stem cells caused significant
decreases in expression of ACTA2 (.alpha.-SMA), CDH2 and Collagen 1
in UIP cells. The effect was more significant than that of HGF.
Moreover, as shown in FIG. 2, co-culture of UIP cells and human
adipose tissue-derived mesenchymal stem cells was found to cause a
decrease in the level of phosphorylated ERK in UIP cells.
[0095] Furthermore, microarray analysis confirmed that the addition
of human adipose tissue-derived mesenchymal stem cells causes an
increase in the expression of PTPRR gene in UIP cells. This
suggested a possibility such that in UIP cells, PTPRR, the
expression of which has been promoted by the effect of human
adipose tissue-derived mesenchymal stem cells, causes
dephosphorylation of phosphorylated ERK, and thus inhibits the
proliferation of cells, such as UIP cells, involving fibrosis,
thereby treating fibrosis.
3. Effects of Adipose Tissue-Derived Mesenchymal Stem Cells on
Fibrosis-Related Gene Expression of Alveolar Epithelial Cell Line
A549 (Example 3)
[0096] An alveolar epithelium-like cell line (A549;
5.0.times.10.sup.4 cells/well) suspended in 1% serum-containing
DMEM was seeded in a 24-well plate, and then the 24-well plate was
placed in a CO.sub.2 incubator (37.degree. C., 5% CO.sub.2) to
start culture. Twelve (12) hours after the start of culture,
epithelial-to-mesenchymal transition was induced at the final
TGF-.beta. concentration of 100 ng/ml. After 1 day of culture,
TGF-.beta. was completely washed out using DMEM, and transwell
inserts (conning, #3460) were set for an ADMSC co-culture group.
Human adipose tissue-derived mesenchymal stem cells
(2.0.times.10.sup.5cells/well) prepared, subcultured and
cryopreserved in the same manner as in Example 1 were suspended in
1% serum-containing DMEM and then the resultant was added to the
transwell inserts. Twenty four (24) hours after the start of
co-culture, RNA of A549 cells was collected, and then the mRNA
expression levels of fibrosis-related genes, ACTA2 (.alpha.-Smooth
muscle actin; .alpha.-SMA), CDH2 (Cadherin 2) and FN1 (Fibronectin
1) were measured by quantitative PCR. The results are shown in FIG.
3.
[0097] As shown in FIG. 3. co-culture of A549 cells and human
adipose tissue-derived mesenchymal stem cells caused significant
decreases in expression of .alpha.-SMA, CDH2 and FN1 in A549 cells.
It was thus revealed that human adipose tissue-derived mesenchymal
stem cells suppress epithelial-to-mesenchymal transition (EMT) that
is induced by addition of TGF-.beta. and thought to be a cause of
fibrosis, and cause decreases in the levels of fibrosis-related
genes, .alpha.-SMA, CDH2 and FN1.
4. Preparation of Fibrosis Model Mice using Severe Combined
Immunodeficient Mice (Example 4)
[0098] UP cells prepared in the same mariner as in Example 2 were
intravenously administered to severe combined immunodeficient mice.
SCID-Beige (CB17. Dg-PrkdescidKystbg-J/CrlCrli) mice (7- to
10-week-old, female, CHARLES RIVER LABORATORIES JAPAN, INC.). Note
that UIP cells were centrifuged to remove medium, 500 .mu.L of
cells (2.times.10.sup.6 cells) suspended in PBS (manufactured by
Wako) was used for the administration. Mice to which only PBS had
been administered were designated as negative control mice.
Sixty-three (63) days after administration of PBS and UIP cell
suspension, pathological sections of the lungs were prepared arid
then subjected to Masson trichrome stain. The results are shown in
FIG. 4. As shown in FIG. 4, in the group to which the UIP cell
suspension had been administered, clear fibrosis was confirmed and
UP-specific lesions similar to those observed in humans were
observed.
5. Therapeutic Effects of ADMSC in the Fibrosis Model Mice (Example
5)
[0099] As in Example 4, LIP cells prepared in the same manner as in
Example 2 were intravenously administered to severe combined
immunodeficient mice, SCID-Beige (CB17,
Dg-PrkdescidKystbg-J/CrlCrli) mice (7- to 10-week-old, female,
CHARLES RIVER LABORATORIES JAPAN, INC.). Thirty-five (35) days
after administration of UP cells, PBS alone (200 .mu.L,
manufactured by Lonza; PBS group) and human adipose tissue-derived
mesenchymal stern cells (200 .mu.L, 1.5.times.10.sup.6 cells; ADMSC
group) prepared and subcultured in the same manner as in Example 1
were intravenously administrated respectively. Sixty-three (63)
days after administration of UIP cells, pathological sections of
the lungs were prepared, subjected to Masson trichrome stain, and
then to histological analysis. Alleviation of fibrosis was observed
in the ADMSC group compared to the PBS group, suggesting that ADMSC
have a therapeutic effect on pulmonary fibrosis.
INDUSTRIAL APPLICABILITY
[0100] According to the present invention, a novel therapeutic
agent for pulmonary fibrosis, a PTPRR expression promotor and a
novel kit for treatment of pulmonary fibrosis can be provided. The
novel therapeutic agent for pulmonary fibrosis and the novel kit
for treatment of pulmonary fibrosis of the present invention can
exhibit significant therapeutic effects on pulmonary fibrosis, for
which currently no effective pharmacotherapy has been
established.
* * * * *